1. Field of the Invention
The present invention relates to an improved method device for differentiating or separating heterogeneous populations of fast and slow acid producing strains of bacteria to produce single strains or clones. In particular, the present invention relates to a method wherein special growth media and conditions are utilized to achieve the differentiation and wherein the differentiated and selected strains are preferably provided as cultures to producers of fermented products.
2. Prior Art
The principal prior art is described in McKay et al, Applied Microbiology Vol 23, pages 1090-1096 (1972); Limsowtin, G. K. and Terzaghi, B. E. (New Zealand Journal of Dairy Science & Technology Volume 11, pages 65 and 66 (1976)), Limsowtin, G. K., et al New Zealand Journal of Dairy Science Technology 13, pages 1 to 8 (1978), R. J. Marshall et al, Dairy Research, Vol 43, pages 449 to 458 (1976) and in Hull, R. R., The Australian Journal of Dairy Technology pages 65 and 66 (June 1977).
McKay et al describe the problem of the loss of lactose fermenting ability in lactic acid producing cultures in a broth medium. A non-milk agar containing bromocresol purple as an indicator is described for separating colonies which produce acid (yellow) from non-acid producing strains (white) under aerobic conditions. There is no attempt at selection of phase insensitive mutants or recognition of the problem. Thus McKay was studying loss of lactose fermentation and used the non-milk agar medium containing an acid-base indicator to detect non-lactose fermenting (lac.sup.-) cells. The selection of lac.sup.+ cells on the McKay medium would not be a worth while approach to isolating cells which would yield fast acid producing cultures in milk. Another important determinant for fast acid production in milk is proteolysis (prt). The McKay medium only distinguishes between lac.sup.+ and lac.sup.- and lac.sup.+ prt.sup.- and lac.sup.+ prt.sup.+ cells appear the same on his medium, yet the former would be slow in milk while the latter fast. The problem not solved by McKay et al is to distinguish between lac.sup.+ prt.sup.- and lac.sup.+ prt.sup.+ cells, particularly since the large majority of slow acid producing variant cells in milk cultures are lac.sup.+ prt.sup.-.
Limsowtin et al (1976) described a glycerophosphate buffered, nonfat milk-based, agar medium (GMA) for the differentiation of fast and slow milk coagulating lactic streptococci. Aerobic (air) growth conditions were used for the growth of the bacteria. The medium has been found to be impractical to use since it produced uncertain differentiation of fast and slow acid producing cultures of certain Streptococcus cremoris or Streptococcus lactis and it was difficult to see white or translucent streptococcal colonies on the white background of this medium. This two strains known to be fast acid producing strains produced colonies which had only a 0.5 mm colony diameter thereby erroneously indicating that they were all slow acid producers. Oblique illumination was used to obtain the published photographs, however visual selection is difficult. Marshall et al describe other phosphate buffered media for the selection process under aerobic conditions where selection is difficult. In selecting starter strains for commercial fermentations, particularly for making fermented dairy products, the method for differentiating and selecting the strains must be completely reliable because of the large volumes of milk or other food being fermented.
Hull describes a method wherein the method of Limsowtin et al can be used by producers of cultures to provide phage resistant, fast acid producing bacterial cultures to producers of fermented dairy products. By this process a portion of the fermented product or a by-product (whey) provides a source for phage which are mixed on the plating medium with the bacteria for differentiation and selection of phage resistant strains. New phage resistant strains are provided to the producers of fermented dairy products and older strains are dropped as phage appear before they have a change to propagate and to vitally infect the older strains. The problem is that the Limsowtin et al method is not reliable or certain enough to make the method suggested by Hull commercially feasible for the culture producers.